Effect And Mechanism Of HBMSC-Exos In Promoting Human Retinal Progenitor Cell Transplantation For Retinal Pigment Degeneration

Retinitis pigmentosa(RP)is a severe blinding eye disease characterized by progressive retinal photoreceptor cell apoptosis.Since denatured photoreceptor cells cannot be regenerated,stem cell transplantation to replace the degenerated or dead cells is the most promising treatment strategy.Previous research by our research group and other research groups showed that human embryo-derived retinal progenitor cells(RPC)transplanted into RP model animals and patient retinas could improve host visual function,but not last long time.In view of the fact that in RP,the pathological microenvironment have important effect on the survival and function of transplanted cells.Hence,we transplanted bone marrow mesenchymal stem cells(BMSC)and RPC into the subretinal space of Royal Colleag of Surgeons(RCS rats,an RP model animal),which can significantly improve the transplantation efficacy.What is the mechanism of the efficacy of combined transplantation?It is not clear.A large number of studies have shown that mesenchymal stem cells(MSC)can secrete a variety of growth factors,extracellular matrix components,and extracellular vesicles,Among of them,exosomes and other nanoscale vesicles play the function similar to their mother cells,which is important in cell to cell information transmission.Interestingly,BMSCs have significant inflammatory regulatory effects.Therefore,we hypothesized that BMSC-derived exosomes might target the microglia cells,the most relevant cells to the inflammatory microenvironment in RP,to improve the survival and different-iation of RPC.In this study,we explored the effects of BMSC exosomes(BMSC-Exos)on microglia and RPC in vitro and in vivo.We hope to provide evidence for promoting RPC transplantation in RP by BMSC-Exos.The main findings are as follows:1.Preparation of hBMSC-Exos and analysis of miRNA differences in exosomes under different conditionsBMSCs were separated and extracted by density gradient centrifugation,then identified with typical BMSC characteristics by flow cytometry and induced differentiation.hBMSC-Exos were obtained by classic ultra-high speed centrifugation.It was observed by transmission electron microscopy as elliptic and circular with doublelayer membrane structure.Nanoparticle Tracking Analysis(NTA)detected particle size distribution between 40-150nm.Western Blot(WB)identified its surface markers CD63,HSP70,CD9 were positive.The levels of the anti-inflammatory factors were increased in BMSCs pretreated by TNF-α.The exosomes under the two conditions were analyzed by miRNA sequencing and the results showed that the expression levels of miR-21-5p were the highest,but the types and numbers of other miRNA molecules had no significant differences.2.Regulation of biological characteristics of microglia cells and hRPC by hBMSCExosImmunocytochemical staining results indicated that BMSC-Exos were endocytosed by microglia.Furthermore,hBMSC-Exos were co-cultured with lipopolysaccharide(LPS)-treated microglia.Additionally,according to the results of miRNA sequencing,miR-21-5p agonists and antagonists,was transfected into microglia,and RT-PCR was used to analyze the changes of inflammatory factor gene expression.The results showed that hBMSC-Exos and agomiR-21-5p could significantly inhibit the expression of inflammatory factors caused by microglial activation,while antagomir-215p reversed this result.After hBMSC-Exos were added to hRPC culture medium,cck8 and cell cycle experiments showed that hBMSC-Exos promoted hRPC proliferation,RT-PCR and immunohistochemistry experiments showed that hBMSC-Exos promoted hRPC differentiation to photoreceptor cells,bipolar cells and ganglion cells,and cell scratches experiments showed that hBMSC-Exos could promote the migration of hRPC.3.Study on the effect of hBMSC-Exos on hRPC transplantation in RCS ratsRCS rats were selected as RP model animals,and hBMSC-Exos and hRPCs were transplanted into the subretinal space of RCS rats.From the FERG test,we found that compared with the PBS,the amplitudes of both a-wave and b-wave in the trans-plantion were significantly increased.Compared with the hRPC alone transplantation group,the hBMSC-Exos+hRPC transplantation began to improve visual function at an early stage and the effect was more significant.In addition,it was observed by retinal section staining that the protective effect of hBMSC-Exos+hRPC group on the outer retinal nuclear layer was superior to that of the hRPC group alone,and the hBMSC-Exos+hRPC transplantation group gave priority to this role after 2w surgery.Better and earlier inhibition of microglial activation was also observed in hBMSC-Exos+hRPC in immunohistochemical experiments.In summary,these data suggest that hBMSC-Exos have a significant effect in promoting hRPC transplantation for RP.There are two aspects to the mechanism,on the one hand,BMSC-Exos reduced the activation of microglia through its cargo of miR-215p,thereby attenuating the inflammatory response and improving the microenvironment of retina for stem cell transplantation;on the other hand,BMSC-Exos facilitate RPC biological characteristics regardless of proliferation,different-iation and migration.In consequence,hBMSC-Exos transplantation can be used as a new strategy for nerve damage repair followed stem cell transplantation.Expected its double roles of immunoregulation and nerve repairs,hBMSC-Exos transplantation might become a new approach for clinical treatment of retinal degenerative diseases.